1. Field of the Invention
The present invention relates to methods for separating particles based upon the migration of particles in response to an electric field.
2. Description of Related Art
By way of background, particles can be manipulated by subjecting them to travelling electric fields. Such travelling fields are produced by applying appropriate voltages to microelectrode arrays of suitable design. The microelectrodes may have the geometrical form of parallel bars, which may be interrupted by spaces to form channels and may be fabricated using standard metal sputtering and photolithographic techniques as described by Price, Burt and Pethig, Biochemica et Biophysica, Vol.964, pp.221-230. Travelling electric fields are generated by applying voltages of suitable frequency and phases to the electrodes as described in "Separation of small particles suspended in liquid by nonuniform travelling field ", by Masuda, Washizu and Iwadare, IEEE Transactions on Industry Applications, Vol.IA-23, pp.474-480. Masuda and his coworkers describe how a series of parallel electrodes (with no channels) supporting a travelling electric field can, in principle, be used to separate particles according to their electrical charge and size (weight) . Masuda et al have not however described a practical demonstration of such a particle separation method.
In a paper entitled "Travelling-wave dielectrophoresis of microparticles" by Hagedorn, Fuhr, Muller and Gimsa (Electrophoresis, Vol.13, pp.49-54) a method is shown for moving dielectric particles, like living cells and artificial objects of microscopic dimensions, over micro-electrode structures and in channels bounded by the electrodes. The travelling field was generated by applying voltages of the same frequency to each electrode, with a 90.degree. phase shift between neighbouring electrodes.
In "Electrokinetic behaviour of colloidal particles in travelling electric fields: Studies using Yeast cells" by Y Huang, X-B Wang and R Pethig J. Phys. D. Appl. Phys. 26 1993 1528-1535, an analysis supported by experiment is made of the "travelling-wave dielectrophoresis" (TWD) effect described by Hagedorn et al (paper cited above). The phenomenological equation ##EQU1##
is developed by Huang et al, to show that the TWD velocity is a function of the square of the particle radius (r), the square of the electric field strength (A(0)), the periodic length of the travelling field (.lambda.), medium viscosity (.eta.) and the imaginary part of the Clausius-Mossotti factor f(.epsilon..sub.p *, .epsilon..sub.m *) defining the dielectric properties of the particle and the suspending medium in terms of their respective complex permittivities .epsilon..sub.p * and .epsilon..sub.m * This equation provides, for the first time, a practical guide for the design of travelling wave electrode systems for the manipulation and separation of particles.
Although the phenomenon in question is usually termed "travelling wave dielectrophoresis", this is something of a misnomer as the force which acts on the particles to produce translational movement is not the dielectrophoresis force but rather that which acts in electrorotation. This force is related to the imaginary component of the polarisability of the particle within its surrounding medium. However, as is discussed in more detail below, particle migration only occurs for travelling wave frequencies which produce negative dielectrophoretic forces on the particle. (Dielectrophoretic forces are related to the real component of the polarisability of the particle within its surrounding medium.) These forces are responsible for lifting the particle away from the electrodes. We accordingly prefer to refer to the phenomenon called previously "travelling wave dielectrophoresis" by the name "travelling wave field migration" (TWFM). As disclosed in WO94/16821 we have established that to obtain TWFM, two separate criteria have to be met. First, a frequency must be selected at which the dielectrophoresis force acting on the particles is negative, i.e. such as to repel the particles from the electrodes. This, we have found requires the real component of the dipole moment induced in the particles to be negative.
Second, the frequency selected has to be such that the imaginary component of the dipole moment induced in the particles is non-zero (whether positive or negative) to produce a force displacing the particles along the array of electrodes.
In all of these previous proposals where particles are separated on the basis of their TWFM behaviour, the particles are caused to migrate at different rates and those migrating faster are separated from those migrating more slowly or not at all. The sample volumes which can be handled are extremely small, being determined by the size of the apparatus. There is no flow of sample material through the separation apparatus.
DE 4127405 and family number U.S. Pat. No. 5,454,472 disclose the use of a travelling wave electrode array of parallel electrodes to draw particles along a path running transversely to the electrodes. Simultaneously, a field applied is from side to side of the electrode array to draw particles into one of two outlet channels (FIG. 2). The separation of the particles is therefore not due to differing travelling wave field migration properties but differing behaviour under the stationary electrophoresis field. The travelling wave field is used merely to produce movement of the particles through the apparatus.
In "Electrostatic Manipulation of Biological Objects" (J. of Electrostatics 25 (1990) 109-123) Washiza describes a cell separator having an inlet and two outlets between which passes a flow of liquid containing cells. Each cell is held by dielectrophoretic attraction by a 1 mH.sub.2 field and is investigated by means which is not described. Based upon the result of the investigation the cell is released by turning off the field and is either passed to a first outlet by the flow or is deflected to the second outlet by reapplication of the field to a second pair of electrodes. This does not involve separating cells according to their differing TWFM characteristics.